Cloning and expression analysis of Mildew Locus O (MLO) genes related to powdery mildew resistance in Vitis pseudoreticulata, and functional characterisation of VpMLO13

In many plants, Mildew Locus O (MLO) genes facilitate infection by pathogens, including Erysiphe necator, the causative agent of powdery mildew (PM) in grapevines. The function of VvMLOs in Vitis vinifera has previously been demonstrated, but the identity and function of VpMLOs in V. pseudoreticulata are not yet clear. Here, we cloned MLO genes that have been implicated in PM susceptibility from the highly PM-resistant V. pseudoreticulata accession ‘Baihe-35-1’ and the PM-susceptible V. vinifera ‘Carinena’. The open reading frames (ORFs) of MLO3, MLO6, and MLO8 differed between ‘Baihe-35-1’ and ‘Carinena’. After inoculation of the leaves with Erysiphe necator, six MLO genes exhibited two distinct expression patterns in ‘Baihe-35-1’ and ‘Carinena’. Consistent with the MLO proteins in other plants, we also found that VpMLO3, VpMLO6, VpMLO11, and VpMLO13 are all localised to the plasma membrane. To further support a role for VpMLOs in susceptibility to PM, we expressed VpMlo13 in the PM-resistant Arabidopsis thaliana MLO2/6/12 triple mutant, which showed that transgenic expression of VpMLO13 restored susceptibility to PM. In addition, a yeast two-hybrid (Y2H) assay showed that VpMLO13 interacted with calmodulin (VpCaM). These results will provide a basis for further investigating the PM resistance mechanisms in V. pseudoreticulata.